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Abstract

99mTc-N4-guanine (99mTc-N4amG) was synthesized and evaluated in this study. Cellular uptake and cellular fraction studies were performed to evaluate the cell penetrating ability. Biodistribution and planar imaging were conducted in breast tumor-bearing rats. Up to 17%ID uptake was observed in cellular uptake study with 40% of 99mTc-N4amG was accumulated in the nucleus. Biodistribution and scintigraphic imaging studies showed increased tumor/muscle count density ratios as a function of time. Our results demonstrate the feasibility of using 99mTc-N4amG in tumor specific imaging.

INTRODUCTION

Cancer cells are transformed from normal cells. During transformation, certain capacities are acquired by cancer cells, including self-sufficiency in growth signals, insensitivity to anti-growth signals, evading apoptosis, limitless replicative potential, sustained angiogenesis and tissue invasive & metastasis(Hanahan and Weinberg 2000). Of these cancer hallmarks, four out of six are cell survival and proliferation-related. In other words, un-controlled cell proliferation can be marked as the most important characteristic of cancer cell.

Modern imaging technologies provide non-invasive, high resolution functional imaging that assists in assessment of cancer cell proliferation. Positron emission tomography (PET) and single photon emission computed tomography (SPECT) are two major molecular imaging modalities in nuclear medicine. With proper imaging probes, both modalities can be used to evaluate tumor growth potential, determine the degree of malignancy, and even provide physicians with an early prediction of treatment response. Much has been done to develop proper imaging probes for tumor proliferation assessment. It has been reported that the uptake of 2′-fluorodeoxyglucose (18F-FDG) correlates with cell proliferation in vitro and in vivo(Okada, Yoshikawa et al. 1992; Higashi, Clavo et al. 1993). Studies performed by various laboratories demonstrated that radiolabeled amino acid analogues may have similar potential to assess cell proliferation as well(Jager, Plaat et al. 2000; McConathy, Martarello et al. 2003). Indeed, both sugars (such as 18F-FDG) and amino acids play important roles in cell proliferation; however, nucleosides are the most essential elements in generation of nucleotides, which are required in cell proliferation. During cell proliferation, DNA must be duplicated. Therefore, uptake and consumption of nucleosides will be much greater in proliferating cells than in resting cells. Hence, radiolabeled nucleoside analogues are potentially the ideal tumor cell proliferative markers.

Development of radiolabeled nucleoside analogues which are involved in DNA/RNA synthesis or incorporated into DNA/RNA has been previously reported(Wright, Taylor et al. 1969; Uesugi, Kaneyasu et al. 1982; Marquez, Tseng et al. 1990; Buck, Halter et al. 2003; Francis, Visvikis et al. 2003). These probes made it possible to assess cell proliferation. For example, 3′-deoxy-3′-18F-fluorothymidine (18F-FLT) is a clinically-tested tracer which images cellular proliferation by involving in DNA synthesis process. Many studies have been conducted to compare the sensitivity and specificity of 18F-FDG and 18F-FLT in different tumor types. For instance, comparison between 18F-FDG and 18F-FLT in glioma shows 18F-FLT exhibits greater sensitivity than 18F-FDG, with higher correlation to proliferation marker, Ki-67(Chen, Cloughesy et al. 2005). Other studies were focused on the potential utility of 18F-FLT to assess treatment response. Data from different studies have shown that 18F-FLT can be used to predict survival rate of patient with glioma after bevacizumab and irinotecan treatment(Chen, Delaloye et al. 2007), define proliferation of tumor treated with cytostatic kinase inhibitor(Waldherr, Mellinghoff et al. 2005), and evaluate dose scheduling(Liu, Kolesar et al. 2008). However, DNA incorporation of 18F-FLT is low, and the chemistry of making 18F-FLT is complex(Buck, Halter et al. 2003; Francis, Visvikis et al. 2003). With such a complex chemistry, the radiochemical yield of 18F-FLT is low, as expected. In addition, 18F can only be obtained from cyclotron, which is inconvenient and expensive. All of these result in limited clinical applications of 18F-FLT. Furthermore, although it has been accepted that the phosphorylation of 18F-FLT by cellular thymidine kinase type 1 (TK1) is the key cellular factor for 18F-FLT uptake, some clinical trials have shown a low or even no correlation between proliferation markers and 18F-FLT tumor uptake(van Westreenen, Cobben et al. 2005; Westerterp, van Westreenen et al. 2005; Linecker, Kermer et al. 2008; Kimura, Yamamoto et al. 2009), indicating that other unknown cellular factors may mediate 18F-FLT uptake. Some of these factors that mediate 18F-FLT uptake include human nucleoside transporters (hNTs)(Paproski, Wuest et al. ; Paproski, Ng et al. 2008).

To overcome the low radiochemical yield and complex synthetic chemistry of 18F-FLT, chelation chemistry plus in-house generator can be used to develop a novel tracer to measure changes in proliferative activity. Using chelation chemistry, radioisotopes can be labeled to a homing agent through a chelator. With this concept, single homing agent-chelator conjugate can be labeled with two or more different radioisotopes for various purposes. For example, 99mTc and 68Ga-labeled compounds can be used for SPECT and PET imaging, respectively; 188Re labeled compounds can be used for radiation therapy. Among all radioisotopes, 99mTc has been preferred to label imaging probes because of its favorable low energy (140 keV vs. 511 keV for 18F), inexpensive isotope cost and capacity for kit product manufacturing. Unlike 18F-labeled compounds that have to be delivered directly from manufacturing site shortly before administration, the cold kit product can be stored and labeled onsite with radioisotopes generated from an in-house generator when needed. Many 99mTc-labeling techniques have been developed. For instance, cyclam (N4), DTPA, DOTA, sulfur colloid and N2S2 have all been labeled with high radiochemical purity and yield(Van Nerom, Bormans et al. 1993; Laissy, Faraggi et al. 1994; Kao, ChangLai et al. 1998; Vriens, Blankenberg et al. 1998; Ohtsuki, Akashi et al. 1999; Canet, Casali et al. 2000; Wu, Chang et al. 2003). Among these chelators, N4 was chosen because it is a closed-ring structure, which is more rigid to stabilize isotopes. Also, the higher lipophilicity of N4 is preferred because it will help molecules to penetrate cell membrane, leading to greater uptake, decrease kidney accumulation and, thereby, reducing renal toxicity.

Nucleosides are constituted by three key elements: 1. The hydroxymethyl group, which is necessary for the phosphorylation of the molecule by cellular kinases to achieve biological activity. 2. The heterocyclic base moiety, which is crucial in the recognition process of the nucleoside through specific hydrogen bonds. 3. The ribose ring, which acts as a spacer between the hydroxymethyl group and the base and keeps them in the correct orientation. A variety of nucleoside analogues have been discovered; many of them have been successfully developed as therapeutic agents in both anticancer and antiviral medicine(Romeo, Chiacchio et al. 2010). There was few chelator-nucleoside conjugates used in tumor imaging, to explore novel nucleoside-based analogs using chelation radiochemistry, we synthesized a guanine analog using N4 as a chelator. In this report, the synthesis and assessment of tumor growth using 99mTc-N4-guanine (99mTc-N4amG) were evaluated.

MATERIALS AND METHODS

Chemicals and Analysis

Proton NMR and 13C NMR analyses were performed using Bruker 300 MHz NMR spectroscope at the NMR core facility in UT MD Anderson Cancer Center (Houston, TX). Mass spectral analyses were conducted at the University of Texas M.D. Anderson Cancer Center (Houston, TX, USA). High resolution Mass spectra (multimode as ion source) were acquired on an Agilent 6200A accurate Mass time of flight (TOF) system (Santa Clara, CA, USA). HPLC analyses were performed using Waters C-18 column (Milford, MA). Two detectors on HPLC, UV and NaI detectors, were used to detect UV absorption and radioactivity, respectively. The UV absorption wave length was 254nm. Mobile system was acetonitrile to water: 8 to 2. Flow rate was 0.5mL per minite. Penciclovir was purchased from LKT Laboratories (St. Paul, MN). All other chemicals were purchased from Sigma-Aldrich (St. Louis, MO). Silica gel coated thin-layer chromatography (TLC) plates were purchased from Whatman (Clifton, NJ, USA).

In vitro cell culture assay

To evaluate whether 99mTc-N4amG can penetrate cell membrane, in vitro cellular uptake study using 99mTc-N4amG and its chelator control (99mTc-N4) was conducted. Cells in 12-well plate (50,000 cells/ well) were incubated with 99mTc-N4amG or 99mTc-N4 at 37 °C for 30 min, 2 hr or 4 hr. Following the incubation period, cells were washed with PBS twice and detached with 0.5mL of trypsin solution. After centrifugation, radioactivity was measured by gamma counter. Data was processed and presented in mean ±S.D.

To determine the distribution pattern of 99mTc-N4amG within the cells, NE–PER extraction reagents were used stepwise to isolate cytoplasmic and nuclear extracts from breast cancer cells. 99mTc-N4amG (0.1mCi) was prepared in the concentration of 0.1 mg/mL. After 20 ml of 99mTc-N4amG (0.02 mg/well, 2 mCi) or 99mTc-N4 (control) was dispensed into each well of 6-well plate, cells (50,000 per well) were incubated at 37 °C for 2 h. Cells were transferred to a 2mL microcentrifuge tube and isolated by centrifugation at 500 g for 2–3 min. 200 μL of ice-cold kit reagent (CER I) was added to the cell pellet in other remaining tubes. Following 10 min incubation, 11 μL of ice cold CER II was added and incubated for an additional 1 min. The tubes were centrifuged for 5 min at maximum speed in a microcentrifuge and the supernatant (cytoplasmic extract) fractions were transferred to other tubes for counting. Insoluble (pellet) fractions, which contain nuclei, were resuspended in 100 μL of ice-cold NER. Following a total of 40 min incubation, the tubes were recentrifuged at a maximum speed for 10 min and the supernatant (nuclear extract) fractions were transferred to other tubes for counting. Insoluble (pellet) fraction, which contains nuclear material, was resuspended in 100 μL of 1_PBS and transferred to other tubes for counting. Samples were counted in a gamma counter.

To further investigate whether different cell cycle phases have different capacity in the uptake of 99mTc-N4-Guan, cell sorting assay was conducted. Cells were sorted into the various phases of cell cycle (G0/G1, S, and G2/M) using fluorescence activated cell sorter (FACS). Sorted cells were suspended in 15mL test tubes (50,000 cells/ tube). 99mTc-N4-Guan or 99mTc-N4 was added, and the cells were incubated at 37 °C for 60 min. Following the incubation period, cells were centrifuged and washed with PBS twice. After final centrifugation, radioactivity was measured by gamma counter. Data was processed and presented in mean ±S.D.

Biodistribution studies

The animals were housed in the University of Texas M.D. Anderson Cancer Center facility. The protocols involving rats and radioisotopes were approved by the M.D. Anderson Animal Use and Care Committee, and Radiation Safety Committee. Female Fischer 344 rats (150–175 g) (Harlan Sprague–Dawley, Inc., Indianapolis, IN) were inoculated subcutaneously in the right leg with breast cancer cells (106 cells/rat) from the breast cell line (known as DMBA-induced breast cancer cell line). Biodistribution studies were performed on day 14 after inoculation. 9 tumor-bearing rats were used and divided into three groups, each group representing a time interval (0.5, 2, and 4 hr, n=3/time point). 20 μCi of 99mTc-N4amG was injected via a lateral tail vein. After administration of the radiotracers, the rats were sacrificed at certain time point and the selected tissues were excised, weighed and counted for radioactivity with a gamma counter. The distribution of radiotracer in each organ was expressed as percentage of the injected dose per gram of tissue (%ID/g). Tumor/ normal tissue count ratios were determined from the corresponding %ID/g values. In statistical analysis, percent of injected dose per gram of tissue weight (%ID/g) and tumor-to-tissue ratios used in biodistribution studies will be presented as means ± SD.

Olinda dosimetry estimation

Human radiation dose estimation for 99mTc-N4amG were computed through Olinda/EXM software (Oakridge, TN)(Stabin, Sparks et al. 2005) from their respective biodistribution data. Olinda inputs were calculated using established method(Wei, Tsao et al. 2008; Tsao, Wang et al. 2011). Briefly, fitted residence time functions were plotted and multiplied by the exponential decay functions (i.e. half-life) for 99mTc. These functions were then integrated analytically to determine the area under the curve (AUC) to yield the residence time of each organ. Mass correction factors were used to account for the different ratios of organ to total body weights in the rat and in humans, and allowed for the scaling of the rat residence times to human residence times. Residence times were then used to calculate target organ absorbed radiation doses with S-value tables for a standard 70 kg male model using the Olinda software package. Each organ dose was computed from the sum of self-dose plus the dose it received from each source organ in the body or from the remainder of the body. The estimated human radiation absorption dose was determined.

Scintigraphic imaging studies

To demonstrate the application of 99mTc-N4amG in breast tumor imaging, three breast tumor-bearing rats were administered with 99mTc-N4amG (300 μCi/rat, 1.0mg physical amount, i.v.) Scintigraphic images, using a gamma camera equipped with low-energy, parallelhole collimator, were obtained after 1, 2 and 4 hr. The standard source used for calibration was 30 μCi of Na99mTcO4. Computer outlined region of interest (counts per pixel) of the tumor and muscle (at the symmetric site) was used to determine tumor-to-muscle count density ratios.

Radio HPLC analysis of 99mTc-N4amG. The spectra showed that the radiochemical purity of 99mTc-N4amG was greater than 95%.

In vitro cell culture assays

High cellular uptake (up to 17%ID at 4 hr) of 99mTc-N4amG in breast tumor cells was observed in cellular uptake study (Fig. 5). The accumulation increased with increase in incubation time suggesting 99mTc-N4amG has tumor targeting potential. Nucleus fraction had 40% uptake of 99mTc-N4amG whereas cytosolic fraction had 60%. Both nucleus and cytosolic fractions in 99mTc-N4amG were higher than 99mTc-N4 (Fig. 6). The data indicated that 99mTc-N4amG, but not 99mTc-N4 can penetrate cell membrane, and about 40% of 99mTc-N4amG will accumulate in cell nucleus. In cell sorting assay, cells in S phase showed highest uptake of 99mTc-N4-Guan followed by G2/M phase (Fig. 7). Compare to chelator-only control (99mTc-N4), 99mTc-N4-Guan showed greater uptake in each cell cycle phases. This data suggested 99mTc-N4-Guan uptake is S phase-related.

in vitro cellular uptake of 99mTc-N4amG. Breast tumor cells (50,000 per each well) were incubated with 2 μCi of 99mTc-N4amG at 37°C at 0.5–4 hr. There was a gradually increased uptake of 99mTc-N4amG in mammary tumor cells. Data...

in vitro cellular fractions of 99mTc-N4amG. After 2ml of radioactive media containing 2 μCi (0.074MBq) of 99mTc-N4amG (0.02 mg/well) or 99mTc-N4 (control) was dispensed into each well of 6-well plate, cellular fractions were isolated by using...

in vitro cell sorting assay of 99mTc-N4amG. Cells were sorted into the various phases of cell cycle (G0/G1, S, and G2/M) using fluorescence activated cell sorter. Sorted cells were suspended in 15mL test tubes (50,000 cells/ tube). 99mTc-N4-Guan or 99m...

Biodistribution studies

In biodistribution studies, there was an increased uptake in tumor-to-blood and tumor-to-muscle ratios in breast tumor-bearing rats. The optimum tumor uptake and tumor-to-muscle ratios were 2–4 hr post-administrations. There was no increased uptake in thyroid and stomach suggesting in vivo stability of 99mTc-N4amG (Table 1). 99mTc-N4amG was excreted through liver and kidneys. Relatively high uptake in Bone and spleen was observed suggesting 99mTc-N4amG could target organs with high proliferation properties.

Olinda dosimetry estimation

The human residence time estimates were applied to the Olinda software to produce radiation dose estimates for 99mTc-N4amG. The Olinda output was multiplied by a proposed human dose of 20mCi for 99mTc-N4amG to yield total absorbed dose from an imaging study (Table 2). Radiation exposure to the whole body, blood forming organs (red marrow, spleen), gonads (testes, ovaries), and effective dose equivalent for the proposed human single dose at 20 mCi fall below the limits of 3 rad annually and 5 rad total. The absorbed dose in all other organs (e.g. kidneys) was below the limits of single dose of 5 rad annually and 15 rad in total. 99mTc-N4amG showed total rad absorbed by each organ was below the proposed annual and total limits.

DISCUSSION

Penciclovir was selected as the lead compound to synthesize N4amG because of four major concerns: 1. Penciclovir is a guanosine analogue which preserves the core guanine structure. Since guanine is one of the main nucleobases in human body, penciclovir will work as an agonist in human body with high affinity to human nucleoside transporters (hNTs) and can be effectively transported into cell(Colledge, Civitico et al. 2000). 2. The rest part of penciclovir is a carbon chain with two hydroxyl groups instead of a ribose (deoxyribose) in guanosine. The carbon chain makes penciclovir resistant to metabolizing enzymes, as a result, the circulation time and half-life of penciclovir in human body will be increased. 3. The two hydroxyl groups play a crucial role in pharmacodynamics. After being transported inside the cell, the hydroxyl groups of penciclovir can be phosphorylated by cellular deoxyguanosine kinase(Colledge, Civitico et al. 2000). Phosphorylated penciclovir will be trapped inside cell and may be further involved in DNA synthesis. Finally, since penciclovir is a clinical medicine with known pharmacokinetics, pharmacodynamics and toxicity, and it is also commercially available, it is favorable for us to use penciclovir as the lead compound.

In our biodistribution data, it showed relatively high uptake in bone and spleen (1.02±0.21 and 1.18±0.15 %ID per gram of tissue weight at 4 hr, respectively.) Since red bone marrow and spleen are blood forming organs, it suggests that 99mTc-N4-Guan can target highly proliferating cells. It is known that penciclovir can penetrate cell membrand through hNTs; furthermore, inside cells, phosphorylated penciclovir can be recognized as dGMP and thereby involved in DNA synthesis(Colledge, Civitico et al. 2000). With the same nucleoside backbone, 99mTc-N4-Guan may work very similarly with penciclovir inside cells. In addition, from our cellular fraction studies, after two hours incubation, 40% of the 99mTc-N4-Guan accumulated in the nucleus fraction indicating 99mTc-N4-Guan is involved in cell nucleus activity. These data suggests that 99mTc-N4-Guan may be a potential cell proliferation marker for future malignant diseases assessment.

In medicine, correct diagnosis is essential to successful treatment. At the present time, 18F-FDG PET is the most commonly-used diagnostic tool in clinical oncology. However, 18F-FDG may not be specific enough due to high uptake in benign tissues affected with infection and/or inflammation, leading to false positive results(Barrington and O’Doherty 2003). Consequently, it is difficult to make the right diagnosis. An alternative method by targeting cell proliferation is currently being developed to avoid these false positives results. Although many radiotracers have been tested for assessment of tumor cell proliferation in vitro and in vivo, no agent has been approved for clinical use in patients yet. It is then desirable to have a novel radiotracer for imaging cell proliferation. Nucleoside-based radiotracers not only penetrate cell membrane through hNTs but also accumulate inside proliferating cells. By using homing agent-chelator conjugates labeled with a generator produced radioisotope, greater benefits could be achieved in terms of utility, chemistry and cost. Clinical data also supports that nucleoside-based imaging agents will have higher tumor specificity than 18F-FDG in many cancers (Buck, Schirrmeister et al. 2002; Yamamoto, Nishiyama et al. 2008). Therefore, 99mTc-N4-guan is anticipated to be more specific than FDG in detecting tumors. Furthermore, N4amG can be labeled with different isotopes for difference modalities (such as 68Ga for PET) or even for therapeutic utility (188Re for radiation therapy). Hence, a novel platform of radiolabeled N4-Guan could be developed for tumor-specific imaging and treatment.

In conclusion, we designed and synthesized a novel nucleoside-based imaging tracer that may target tumor cell proliferation, for which good imaging agents are currently not available. Our results demonstrated that N4-Guan can be synthesized in an appropriate yield and it can be labeled with 99mTc in high radiochemical yield and purity. In vitro and in vivo biological evaluations of 99mTc-N4-Guan showed it is feasible to 99mTc-N4amG in tumor specific imaging.

Schematic of N4amG synthesis. Briefly, one of the hydroxyl groups of penciclovir was selectively protected by a bulky protection group. The unprotected hydroxyl group was converted to a highly reactive leaving group (tosyl group) and then reacted with...